The field of proteomics is an expanding industry which seeks to understand biological systems at the molecular level. Using advanced analytical instrumentation, which includes mass spectrometry (MS) and separation methods such as electrophoresis and liquid chromatography (LC), scientists endeavor to perform a complete characterization of all proteins expressed by an organism. Proteomics has made significant advances in the medical field, establishing a reputation as a leading technology for drug discovery and disease diagnosis. Once considered a tool for chemists, today's analytical instruments are not only more sensitive, but their more robust operation is bringing such platforms to a greater range of end users, including molecular biologists and clinicians.
Proteomics relies heavily on effective and robust sample preparation methods for all steps of analysis to obtain useful quality information. A diverse set of approaches are available to isolate and manipulate proteins ahead of separation (i.e., electrophoresis/LC) and analysis (e.g., MS) to remove the interfering sample components from the protein which may be naturally present in a biological matrix (e.g., lipids, nucleic acids, salts) or are otherwise added to the sample to assist with protein solubilization, preservation, derivatization, or separation (e.g., buffers, detergents, chemical reagents). Techniques such as solvent induced protein precipitation, which use solvents such as acetone or chloroform/methanol to cause the protein sample to form an insoluble pellet, are capable of removing the majority of contaminating species while retaining the protein sample with high recovery. The protocols for solvent precipitation are well established, although they are considered difficult to perform with high reproducibility, requiring a steady hand and considerable patience as they are time consuming to perform for the large sets of samples commonly handled in a proteomics experiments. Even with its drawbacks, solvent precipitation remains a popular method of protein cleanup due to its effective removal of a wide range of contaminates and high recovery of target protein.
No product currently exists to facilitate the cleanup of protein samples by solvent precipitation. The products which are available generally make use of simple membrane filters to purify samples. These remove large particulates from a biological sample (e.g., when using suction filters) but are not capable of meeting all the needs to improving the solvent precipitation protocol. Membranes having permeability to molecules over a specific mass range are available and include simple dialysis membranes and spin-type molecular weight cutoff filers (Millipore's Amicon™ filters). While widely used, these strategies result in lower product yield. Moreover, removing interfering components may be difficult without extensive washing, which results in long processing times.
Numerous products are available for solid phase extraction (SPE). These methods differ from the precipitation method in that the samples are all kept in solution prior to being adsorbed to a solid support; extraction cartridges usually contain SPE material which is selective to a particular group of molecules either by their physical or chemical properties. These materials may be selective for either the proteins, or the contaminants. For example, the ZipTip™ (Millipore) is now a ‘classic’ approach designed to manipulate small volumes of sample and maintain high protein recovery. Spin filters containing SPE material are also available from various companies which enable SPE to be multiplexed (limited only to the capacity of the centrifuge). Liquid chromatography is a fully automated version of SPE, although this requires expensive instrumentation which becomes tied up in a low-throughput fashion to process samples in series. While high performance liquid chromatography produces a very good purity product and acceptable recovery, this is at high initial instrument cost and a relatively high per-sample time (about 1 hour).
Other methods are available to separate proteins from detergents. These include purification kits available from Pierce, which use selective adsorption of the detergent, and can be used for acceptable purity and relatively fast (10 min) process time. These kits do not separate other impurities, and can have low analyte (protein) recovery. Protein precipitation kits which differ from the standard solvent precipitation methods are available, in which proprietary reagents/solvents are added to cause protein to precipitate. They have acceptable to high recovery, but they require a skilled user and the overall purity is unknown owing to the proprietary reagents involved.
Reviewing the presented methods for the removal of contaminants to generate a pure protein sample with high recovery, there still remains a need for a strategy that can produce purified proteins with broad applicability and with superior purity, recovery, reliability, and speed.